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The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiationᰔ† Diego Pasini,1 Adrian P

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The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiationᰔ† Diego Pasini,1 Adrian P MOLECULAR AND CELLULAR BIOLOGY, May 2007, p. 3769–3779 Vol. 27, No. 10 0270-7306/07/$08.00ϩ0 doi:10.1128/MCB.01432-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved. The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiationᰔ† Diego Pasini,1 Adrian P. Bracken,1 Jacob B. Hansen,2 Manuela Capillo,3,4 and Kristian Helin1* Centre for Epigenetics and BRIC, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark1; Department of Biomedical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark2; Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy3; and Institute of Molecular Oncology of the Italian Foundation for Cancer Research, Via Adamello 16, 20139 Milan, Italy4 Received 3 August 2006/Returned for modification 10 October 2006/Accepted 22 February 2007 Downloaded from Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12؊/؊ mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12؊/؊ ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12؊/؊ ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always http://mcb.asm.org/ sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation. The evolutionarily conserved Polycomb group (PcG) pro- the molecular mechanisms that control cell fate decisions dur- teins silence gene expression through the formation of multi- ing development. protein complexes. The Polycomb repressive complex 2 Cell fate decisions start taking place before embryo implan- (PRC2) contains the PcG proteins EZH2, EED, and SUZ12 tation, and ICM pluripotency is already lost during embryo and catalyzes the di- and trimethylation of lysine 27 of histone gastrulation (ϳE7.5). Cell type specifications occur through on September 4, 2018 by guest H3 (H3K27me2 and H3K27me3, respectively) (8, 12, 19, 28). It the establishment of specific gene expression programs that has been suggested that this modification serves as a “docking- require epigenetic-dependent transcriptional regulation (2). site” for the PRC1 complex and is required for maintaining Epigenetic control of transcription involves modifications of transcriptional repression (8, 19). The three PcG subunits of both DNA and histones, and the factors that can “write” and PRC2 are all essential for embryonic development, and “read” these modifications play a critical role during develop- Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Eed , Ezh2 , and Suz12 embryos all display severe ment (22). defects during gastrulation (15, 30, 32). SUZ12 is required for Consistent with this model of development, it has not been Ϫ/Ϫ PRC2 enzymatic activity, and Suz12 embryos die around 7 possible to establish Ezh2Ϫ/Ϫ ES cell lines in tissue culture days postcoitus (32). Loss of Suz12 leads to global loss of (30). In contrast, EedϪ/Ϫ ES cells can be expanded in tissue H3K27me3 and to destabilization of Ezh2 (9, 32). These re- culture even though the cells lack global levels of H3K27 meth- sults demonstrate the critical role of Suz12 and PRC2 in reg- ylation (26). EedϪ/Ϫ ES cells have an increased expression of ulating normal development and suggest that PRC2 controls differentiation-specific genes, and the cells “tend” to lose plu- the expression of genes essential for early embryogenesis. ripotency (4). The discrepancy between the phenotypes of Embryonic stem (ES) cells isolated from the inner cell mass Ϫ Ϫ Ϫ Ϫ Ezh2 / and Eed / ES cells suggests that Ezh2 could have (ICM) of preimplantation embryos (embryonic day 3.5 [E3.5]) independent functions that do not involve H3K27 methylation. are immortal and pluripotent. Their self-renewal requires leu- In addition, recent reports have shown that both PRC2 and the kemia inhibitory factor (LIF)-dependent Stat3 activation as PRC1 are required to maintain the repression of differentia- well as the expression of ES cell-specific transcription factors tion-specific genes in mouse and humans ES cells as well as in like Nanog and Oct4 (2). ES cells can give rise to all the somatic cells of an organism and can be differentiated in vitro human embryonic lung fibroblasts (TIG3) (4, 5, 21). to all cell types. They are therefore attractive as a tool to study Here we describe the role of Suz12 in ES cell proliferation and differentiation. We show that Suz12 is essential for proper differentiation, but not proliferation, of ES cells. * Corresponding author. Mailing address: Centre for Epigenetics Importantly, we show that different mechanisms of PcG and BRIC, University of Copenhagen, Ole Maaløes Vej 5, 2200 Co- transcriptional regulation exist during development. This penhagen, Denmark. E-mail: [email protected]. Phone: 45 3532 involves active recruitment of PcGs to repress gene expres- 5666. Fax: 45 3532 5669. E-mail: [email protected]. sion; however, we also demonstrate that increased levels of † Supplemental material for this article may be found at http://mcb .asm.org/. H3K27me3 and PcG binding can also correlate with the ᰔ Published ahead of print on 5 March 2007. activation of gene expression. 3769 3770 PASINI ET AL. MOL.CELL.BIOL. MATERIALS AND METHODS RESULTS Derivation of ES cells, culture, genotype, and sex determination. Blastocysts ؊/؊ ϩ Ϫ Establishment of Suz12 ES cells. To study the role of were isolated from the uterus of superovulated pregnant Suz12 / female mice at 3.5 days postcoitus in M16 medium (Sigma). Single blastocysts were cultured Suz12 in ES cell proliferation, we attempted to derive ES cells Ϫ/Ϫ on 0.5% gelatin-coated plates in Glasgow medium (Sigma) supplemented with from the ICM of Suz12 blastocysts. ICM outgrowths iso- glutamine (Gibco), nonessential amino acids (Gibco), sodium pyruvate (Gibco), lated from blastocysts at E3.5 were expanded in tissue culture 50 ␮M ␤-mercaptoethanol–phosphate-buffered saline (PBS; Gibco), and 20% in the presence of LIF. Genotypes of the individual clones ES-cell-tested fetal bovine serum (HyClone) in the presence of 2,000 U/ml of showed that both female and male Suz12Ϫ/Ϫ ES cells could be LIF (ESGRO). ICM outgrowths were expanded and kept in culture in the same Ϫ/Ϫ basal medium with 10% fetal bovine serum and 1,000 U/ml of LIF. Genotyping derived and maintained in culture (Fig. 1A). Suz12 ES cells of single clones was performed as described previously (32). Sex was determined do not display any morphological differences compared to by PCR using the following primers: Xist forward, 5Ј-GCTTTGTTTCACTTTC wild-type and Suz12ϩ/Ϫ cells (Fig. 1A and B, top panels). TCTGGTGC-3Ј; Xist reverse, 5Ј-ATTCTGGACCTATTGGGAAG-GGGC-3Ј; Moreover, the proliferation rate of Suz12Ϫ/Ϫ ES cells did not Ј Ј Ј Sry forward, 5 -GCATTTATGGTGTGGTCCCGTG-3 ; and Sry reverse, 5 -CC differ significantly from wild-type and Suz12ϩ/Ϫ cells (data not Ј Downloaded from AGTCTTGCCTGTATGTGATGG-3 . Ϫ/Ϫ ES cell karyotype and immunostaining. Growing ES cells were treated with shown). Importantly, Suz12 ES cells express normal levels Colticin (10 ␮l/ml; Gibco) for 1 h. Cells were harvested, incubated for 18 min in of the ES cell markers Oct4 and Nanog (Fig. 1C) and have a hypotonic solution (75 mM KCl), and subsequently fixed in fixative solution (1 normal karyotype (Fig. 1D). Consistent with the requirement volume of acetic acid in 3 volumes of methanol). Metaphases were allowed to dry for Suz12 to stabilize Ezh2, Suz12Ϫ/Ϫ ES cells have reduced on slides overnight at 37°C and were stained with DAPI (4Ј,6Ј-diamidino-2- levels of Ezh2 (Fig. 1B). In order to further characterize the phenylindole; Sigma). For immunostaining, ES cells were cultured in normal ES Ϫ/Ϫ cell medium on mitotically inactivated mouse embryonic fibroblasts (MEFs). Suz12 ES cells, we stained for the expression of Oct4 and Cells were fixed for 10 min in 4% buffered formaldehyde and stained with the Nanog. Consistent with the expression data presented in Fig. antibodies indicated in the figure legends in the presence of 10% serum for 1 h 1C, Suz12ϩ/Ϫ and Suz12Ϫ/Ϫ ES cells but not feeder cells ex- in a humid chamber. pressed high levels of both Oct4 and Nanog (Fig. 2A and B), http://mcb.asm.org/ Antibodies. Immunoblotting and immunostaining were performed with the Ϫ Ϫ demonstrating that Suz12 / ES cells present normal ES cell following antibodies: rabbit anti-Suz12, anti-H3K27me3, anti-H3K27me1 (where me1 indicates monomethylation), anti-H3K9me3, anti-H3K9me2, and anti- features. H3K4me2 from Upstate; rabbit anti ␤-tubulin from Santa Cruz; rabbit anti- Suz12 is required for di- and trimethylation of H3K27 in ES H3K27me2 (33); mouse anti-EZH2 BD43 (32); mouse anti-H3K27me2/me3 cells. To determine if Suz12 is
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